A visual detection method for foodborne Listeria monocytogenes using a ratiometric fluorescent probe

Abstract

The present invention discloses a method for visual detection of foodborne Listeria monocytogenes using a ratio fluorescent probe, and belongs to the technical field of food safety. The steps of visual detection are as follows: (1) preparing a food solution; (2) preparing a liquid to be detected, mixing the food solution with a ratio fluorescent probe in proportion, incubating, and obtaining a liquid to be detected; (3) detecting the liquid to be detected, measuring the image of the liquid to be detected under an excitation wavelength of 285nm in the dark by an RGB imager, reading the RGB color value of the image, selecting the ratio between the B value and the G value in the read RGB color value as the ordinate, and taking the logarithm of the bacterial concentration of the bacteria-containing detection liquid as the abscissa, drawing a standard curve, and obtaining the correlation coefficient and the detection limit according to the linear regression equation. The linear fitting coefficient of the detection method is 0.98945, and the detection limit is as low as 6.31CFU / mL. The true concentration of Listeria monocytogenes can be quickly obtained at the food sample detection site.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and in particular relates to a method for visualizing detection of foodborne Listeria monocytogenes using a ratio fluorescent probe. Background Art

[0002] Foodborne diseases are common human diseases that cause infection or poisoning by ingesting food and allowing pathogenic factors to enter the body. They cause serious public health burdens worldwide and have adverse effects on economic and social development. According to data from the World Health Organization from 2020 to 2022, about 550 million to 600 million people worldwide become ill each year from eating contaminated food, of which about 230,000 die. Foodborne pathogens are a major cause of foodborne diseases, among which Listeria monocytogenes is a common pathogen. It is widely present in the environment, food, humans and animal hosts. It can survive under extreme conditions and cause food contamination. Eating food contaminated by this bacterium can cause listeriosis, with a mortality rate of up to 20%-30%, which poses a great threat to public health safety. For the detection of common pathogens, traditional culture methods are still recognized and reliable methods, but they require multiple steps such as bacterial isolation and culture, morphological observation, and biochemical identification, which are relatively cumbersome; conventional polymerase chain reaction (PCR) methods are limited by the long detection time. The ELISA kit based on immunological technology is expensive and difficult to be promoted and used on an industrial scale. Therefore, it is urgent to develop simple, rapid, highly sensitive and accurate strategies and analytical methods for real-time detection of Listeria monocytogenes in food and environment. The detection method of emerging fluorescent biological probes is widely used in the determination of target substances because of its advantages such as low cost, easy operation, fast response and high stability. Among them, fluorescence resonance energy transfer (FRET) is a non-radiative energy transition that transfers donor excited state energy to acceptor excited state through intermolecular electric dipole interaction, and is widely used in fluorescent probe detection technology. At present, the main fluorescent markers used as detection probes include organic fluorescent dyes and quantum dots. However, conventional fluorescent instruments are currently used for detection, which are heavy and have high instrument maintenance costs, making it difficult to effectively detect food samples on site.

[0003] With the rapid development of intelligent detection in recent years, image processing technology has played a huge role in the field of harmful substance concentration detection. However, the technology currently has disadvantages such as a single detection mode and the need for manual analysis, a relatively low scope of application, and high difficulty in data analysis. Summary of the invention

[0004] In view of the problems that the existing detection technology of Listeria monocytogenes in food has great detection difficulty and long time consumption, the present invention provides a visual detection method for foodborne Listeria monocytogenes using a ratio fluorescent probe.

[0005] The operation steps of visual detection of foodborne Listeria monocytogenes by a ratiometric fluorescent probe are as follows:

[0006] (1) Preparation of food solution

[0007] Artificially inoculating Listeria monocytogenes into the food to be tested to prepare a food solution; the food to be tested is tap water, orange juice, milk, cooked beef, and lettuce, and the food solution is tap water solution, orange juice solution, milk solution, cooked beef solution, and lettuce solution;

[0008] (2) Preparation of test solution

[0009] Mix the ratio fluorescent probe with the food solution obtained in step (1) at a volume ratio of 1:1, and incubate for 25 minutes to obtain a test solution;

[0010] The ratio fluorescent probe is prepared by uniformly mixing a first fluorescent probe as an energy donor and a second fluorescent probe as an energy acceptor in a volume ratio of 1:1;

[0011] The first fluorescent probe is vancomycin-modified carbon quantum dots;

[0012] The second fluorescent probe is aptamer-modified silicon nanoparticles;

[0013] The DNA sequence of the aptamer is shown in SEQ ID No: 1, and the 5' end of the aptamer is modified with an amino group;

[0014] In the ratiometric fluorescent probe, the fluorescence emission wavelength of the vancomycin-modified carbon quantum dots is 458 nm, and the fluorescence emission wavelength of the aptamer-modified silicon nanoparticles is 488 nm;

[0015] (3) Test the liquid to be tested

[0016] The liquid to be tested obtained in step (2) is measured by a red, green and blue three-color imager under an excitation wavelength of 285 nm to generate an image of the liquid to be tested in the dark, and the red, green and blue values ​​of the image are read;

[0017] The ratio between the blue value and the green value in the red, green and blue values ​​is selected as the ordinate, and the logarithm of the bacterial concentration of the test solution is selected as the abscissa to draw a standard curve, and the correlation coefficient and the detection limit are obtained according to the linear regression equation;

[0018] The linear fitting coefficient of the tap water is 0.98211, and the detection limit is 6.31 CFU / mL. For the tap water solution, when the ratio between the blue value and the green value in the red, green and blue values ​​read is greater than 1.85, Listeria monocytogenes is not detected in the tap water; when the ratio is less than 1.85, Listeria monocytogenes is contained in the tap water, and the concentration of Listeria monocytogenes in the tap water solution can be obtained by substituting the ratio into the linear regression equation;

[0019] The linear fitting coefficient of the orange juice is 0.98090, and the detection limit is 8.51 CFU / mL. For the orange juice solution, when the ratio between the blue value and the green value in the red, green and blue values ​​read is greater than 1.91, Listeria monocytogenes is not detected in the orange juice; when the ratio is less than 1.91, Listeria monocytogenes is contained in the orange juice, and the concentration of Listeria monocytogenes in the orange juice solution can be obtained by substituting the ratio into the linear regression equation.

[0020] The linear fitting coefficient of the milk is 0.98242, and the detection limit is 8.93 CFU / mL. For the milk solution, when the ratio between the blue value and the green value in the red, green and blue values ​​read is greater than 1.90, Listeria monocytogenes is not detected in the milk; when the ratio is less than 1.90, Listeria monocytogenes is contained in the milk, and the concentration of Listeria monocytogenes in the milk solution can be obtained by substituting the ratio into the linear regression equation.

[0021] The linear fitting coefficient of the cooked beef is 0.98945, and the detection limit is 15.09 CFU / mL; for the cooked beef solution, when the ratio between the blue value and the green value in the red, green and blue values ​​read is greater than 1.97, Listeria monocytogenes is not detected in the cooked beef; when the ratio is less than 1.97, Listeria monocytogenes is contained in the cooked beef, and the concentration of Listeria monocytogenes in the cooked beef solution can be obtained by substituting the ratio into the linear regression equation;

[0022] The linear fitting coefficient of the lettuce is 0.97770, and its detection limit is 9.60 CFU / mL; for the milk solution, when the ratio between the blue value and the green value in the read red, green and blue values ​​is greater than 2.03, Listeria monocytogenes is not detected in the milk; when the ratio is less than 2.03, Listeria monocytogenes is contained in the milk, and the concentration of Listeria monocytogenes in the milk solution can be obtained by substituting the ratio into the linear regression equation.

[0023] The technical solution is further defined as follows:

[0024] In step (1), the steps for preparing the tap water solution, orange juice solution or milk solution are as follows:

[0025] Listeria monocytogenes was artificially inoculated into 1 mL of tap water, orange juice or milk, and the final concentration of Listeria monocytogenes was 3.9*101 CFU / mL, 3.9*10 2 CFU / mL, 3.9*10 3 CFU / mL, 3.9*10 4 CFU / mL, 3.9*10 5 CFU / mL, 3.9*10 6 CFU / mL, 3.9*10 7 CFU / mL; centrifuge the seven solutions at 5000×g for 5 min respectively and discard the supernatant; resuspend the precipitates of the seven solutions with the supernatant discarded in 1 mL of phosphate buffered saline respectively to obtain seven tap water solutions or orange juice solutions or milk solutions.

[0026] In step (1), the steps for preparing the cooked beef solution or the lettuce solution are as follows:

[0027] (1.1) Inoculate cooked beef or lettuce with Listeria monocytogenes at an inoculum of 3.9*10 8 CFU / g, after standing for 2 h at room temperature, inoculated cooked beef or inoculated lettuce were obtained;

[0028] (1.2) Cut the inoculated cooked beef into small pieces or cut the inoculated lettuce into pieces;

[0029] (1.3) In a sterilization bag, mix 25 g of inoculated cooked beef cut into small pieces or 25 g of inoculated lettuce cut into pieces with 25 mL of phosphate buffered saline solution and homogenize for 2 minutes to prepare a phosphate buffered saline mixture. Let the mixture stand for 5 minutes and take the supernatant. The final concentration of Listeria monocytogenes in the supernatant was adjusted to 3.9*10 1 CFU / mL, 3.9*10 2 CFU / mL, 3.9*10 3 CFU / mL, 3.9*10 4 CFU / mL, 3.9*10 5 CFU / mL, 3.9*10 6 CFU / mL, 3.9*10 7 CFU / mL is seven parts solution, that is, seven parts cooked beef solution or seven parts lettuce solution.

[0030] In step (2), the preparation operation of the ratiometric fluorescent probe is as follows:

[0031] (2.1) Preparation of activation mixture

[0032] Mix 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide in a volume ratio of 1:1 to obtain an activated mixed solution;

[0033] (2.2) Preparation of the first fluorescent probe

[0034] Guanidine hydrochloride and ultrapure water were mixed and dissolved at a mass volume ratio of 12 mg: 2 mL to obtain a mixed solution; the mixed solution was transferred to a reactor, the reaction temperature was 200°C, the reaction time was 8 hours, and the reaction solution was cooled to room temperature to obtain a reaction solution; the reaction solution was centrifuged at a speed of 8000 rpm and a temperature of 4°C for 20 minutes, and the supernatant was collected; the supernatant was mixed with the activation mixed solution at a volume ratio of 2:1, and incubated at room temperature for 25 minutes; a 50 uM vancomycin solution was added, the amount of the vancomycin solution added was 0.006 times the volume of the supernatant, and the reaction was incubated on a shaker at a speed of 180 rpm and room temperature for 2.5 hours to obtain a first fluorescent probe;

[0035] (2.3) Preparation of the second fluorescent probe

[0036] A sodium ascorbate solution with a molar concentration of 100 mM and 3-aminopropyltrimethoxysilane were mixed in a volume ratio of 1:1, and eight times the volume of ultrapure water was added and stirred for 80 minutes; dialyzed with a 500Da dialysis bag for 18 hours to obtain a retentate solution; the retentate solution was mixed with the activation mixed solution in a volume ratio of 2:1, and incubated at room temperature for 30 minutes; an aptamer solution with a concentration of 100 uM was added, and the amount of the aptamer solution added was 0.03 times the volume of the retentate solution; at a rotation speed of 180 rpm and room temperature, the shaker was incubated for 2.5 hours to obtain a second fluorescent probe;

[0037] (2.4) Preparation of ratiometric fluorescent probe

[0038] The first fluorescent probe and the second fluorescent probe are mixed at a volume ratio of 1:1 to prepare a ratio fluorescent probe;

[0039] When the fluorescence emission wavelength of the carbon quantum dots shifts from 452nm to 458nm, it indicates that vancomycin in the ratio fluorescence probe is successfully modified onto the carbon quantum dots. When the fluorescence emission wavelength of the silicon nanoparticles shifts from 500nm to 488nm, it indicates that the aptamer in the ratio fluorescence probe is successfully modified onto the silicon nanoparticles.

[0040] The beneficial technical effects of the present invention are embodied in the following aspects:

[0041] 1. The detection method of the present invention only requires an effective combination of two existing small commercial devices, and rapid analysis can be performed by detecting the red, green and blue values.

[0042] It has been verified that the visualization detection method of the present invention has a high degree of linear fit (correlation coefficient as high as 0.98211) and a low detection limit (as low as 6.31 CFU / mL) when applied to tap water detection. The linear fit degree of similar detection methods in the current prior art generally ranges from 0.89 to 0.94; at the same time, compared with traditional portable detection technologies (ELISA kit method, portable fluorescence spectrometer), their detection time varies from 6h to 24h, while the visualization detection method combined with ratio fluorescence probe has a lower detection time, which only takes 25min to 30min. For example, the traditional enzyme-linked immunosorbent assay kit method is costly and requires the use of professional bulky equipment for detection and analysis. It is impossible to achieve good quantitative detection on site, and the application environment is limited. In addition, the present invention has serious environmental interference for common food samples such as orange juice samples, milk samples, cooked beef samples, and lettuce samples, but the correlation coefficient is above 0.9777, which meets the higher quantitative detection requirements. At the same time, the highest detection limit is only 5.09 CFU / mL.

[0043] 2. After testing, the linear regression equation established in the tap water environment can be directly applied to the detection of LB broth samples. There is no need to re-cultivate bacteria to establish a standard curve. Just apply the established linear regression equation directly, which saves time and is suitable for quickly estimating the initial concentration of Listeria monocytogenes in food samples under emergency conditions. For example, in the LB broth sample, in the environment where Listeria monocytogenes was artificially inoculated in the LB broth, the concentration of Listeria monocytogenes was measured to be 3.83*10 6 CFU / mL, almost the same as its actual concentration (3.9*10 6 CFU / mL) is close. It can be seen that even if the corresponding standard curve and linear regression equation in the LB broth sample environment are not established, the standard curve and linear regression equation established in the tap water solution environment can still have good versatility. It can be seen that the method of the present application has good versatility and can be quickly applied to different sample detections.

[0044] 3. According to statistics, the present invention can achieve rapid detection of Listeria monocytogenes within 30 minutes. With the help of a lightweight image detection device, it only needs to detect the red, green and blue values ​​of the image without the need to carry professional fluorescence spectrum equipment, which greatly improves the detection efficiency of Listeria monocytogenes in various food samples. At the same time, it reduces the requirements for instruments and equipment and the quality of laboratory personnel, and provides strong technical support for the early diagnosis, disease monitoring and precise targeted elimination of foodborne Listeria monocytogenes. In the existing traditional methods, it is necessary to carry a bulky polymerase chain reaction nucleic acid amplification instrument or an expensive enzyme-linked immunosorbent assay kit, which makes it impossible to achieve on-site detection, hindering the application of on-site detection technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is the detection image and standard curve diagram of the tap water solution in the present invention.

[0046] Figure 2 It is the detection image and standard curve diagram of the orange juice sample in the present invention.

[0047] Figure 3 It is the detection image and standard curve diagram of the milk sample in the present invention.

[0048] Figure 4 It is the detection image and standard curve diagram of the cooked beef sample in the present invention.

[0049] Figure 5 It is the detection image and standard curve diagram of the lettuce sample in the present invention. DETAILED DESCRIPTION

[0050] The present invention is further described below in conjunction with specific embodiments.

[0051] Unless otherwise defined, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0052] The raw materials used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified; the quantitative tests in the following examples are all repeated three times, and the results are averaged; the % in the following examples are all mass percentages unless otherwise specified.

[0053] In the following examples, the aptamers used were purchased from Shanghai Bioengineering Co., Ltd.; 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, guanidine hydrochloride, vancomycin, sodium ascorbate and 3-aminopropyltrimethoxysilane used were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the 500Da dialysis bag used was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; unless otherwise specified, other raw materials used were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0054] The phosphate buffered saline solution used in this application is a sterile phosphate buffered saline solution with a pH value of 7.4 and a 0.1 M concentration.

[0055] All bacterial strains used in this application were purchased from the American Type Culture Collection (ATCC), among which Listeria monocytogenes is numbered ATCC 43251. The above strains were purchased through overseas agent Hefei Xiyue Biotechnology Co., Ltd.

[0056] The instruments, equipment, raw materials, reagents or method steps used in this application are ensured to be processed under sterile conditions.

[0057] The instruments, equipment, raw materials, reagents or method steps not mentioned in this application are conventional or well-known technical methods for those skilled in the art and are not described in detail in this application. Please refer to the following paper materials (yuwei Ren, Lulu Cao, Xiyan Zhang, Rui Jiao, Dexin Ou, Yang Wang, Danfeng Zhang, Yizhong Shen, Na Ling, Yingwang Ye, A novel fluorescence resonance energy transfer (FRET)-based paper sensor with smartphone for quantitative detection of Vibrio parahaemolyticus, Food Control, Volume 145, 2023, 109412, ISSN 0956-7135, https: / / doi.org / 10.1016 / j.foodcont.2022.109412.).

[0058] Example 1

[0059] The preparation steps of the ratiometric fluorescent probe are as follows:

[0060] (1) Preparation of activation mixture

[0061] Mix 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide in a volume ratio of 1:1, vortex mix in a vortex machine to obtain an activated mixed solution, and store it in a refrigerator at 4°C for later use;

[0062] (2) Preparation of the first fluorescent probe

[0063] Guanidine hydrochloride and ultrapure water were mixed and dissolved at a mass volume ratio of 12 mg: 2 mL, and a vortex machine was used for sufficient shaking to obtain a mixed solution; the mixed solution was transferred to a hydrothermal synthesis reactor with a polytetrafluoroethylene liner for reaction, and the temperature was raised to 200°C in an oven for reaction, and the reaction time was controlled to be 8 hours to obtain a reaction solution, and the reaction solution was cooled to room temperature after the reaction; the reaction solution was centrifuged at 8000 rpm and 4°C for 20 minutes, and the supernatant was collected; 500 uL of the supernatant and 250 uL of the activated mixed solution were respectively taken at a volume ratio of 2:1, and incubated at 180 rpm at room temperature for 25 minutes using a shaker; 3.2 uL of a 50 uM vancomycin solution was added, wherein the volume of the vancomycin solution was 0.006 times the volume of the supernatant, and the shaker was incubated at 180 rpm and room temperature for 2.5 hours to obtain a first fluorescent probe;

[0064] (3) Preparation of the second fluorescent probe

[0065] A 100 mM sodium ascorbate solution and 3-aminopropyltrimethoxysilane were mixed in a volume ratio of 1:1, and eight times the volume of ultrapure water were added and stirred for 80 minutes;

[0066] The retentate was dialyzed for 18 h using a 500 Da dialysis bag and diluted fifteen times;

[0067] The retention liquid was mixed with the activation mixture in step (1) at a volume ratio of 2:1, and incubated at room temperature for 30 minutes; 16uL of 100uM aptamer solution (aptamer sequence is shown in SEQ ID NO.1) was added, and the amount of aptamer solution added was 0.03 times the volume of the retention liquid; the mixture was incubated on a shaker at a speed of 180rpm and room temperature for 2.5 hours to obtain a second fluorescent probe;

[0068] (4) Preparation of ratiometric fluorescent probes

[0069] 50uL of the first fluorescent probe and 50uL of the second fluorescent probe were mixed at a volume ratio of 1:1 to prepare a ratio fluorescent probe;

[0070] When the fluorescence emission wavelength of the carbon quantum dots shifts from 452nm to 458nm, it indicates that vancomycin in the ratio fluorescence probe is successfully modified onto the carbon quantum dots. When the fluorescence emission wavelength of the silicon nanoparticles shifts from 500nm to 488nm, it indicates that the aptamer in the ratio fluorescence probe is successfully modified onto the silicon nanoparticles.

[0071] Example 2 (the food tested is tap water)

[0072] The tap water in this embodiment 2 was taken from the tap water pipe of the school toilet, and the temperature of the tap water was 6°C.

[0073] The steps for visual detection of foodborne Listeria monocytogenes using ratiometric fluorescent probes are as follows:

[0074] (1) Preparation of tap water solution

[0075] Listeria monocytogenes was artificially inoculated in 1 mL of tap water, and the final concentrations of Listeria monocytogenes were 3.9*10 1 CFU / mL, 3.9*10 2 CFU / mL, 3.9*10 3 CFU / mL, 3.9*10 4 CFU / mL, 3.9*10 5 CFU / mL, 3.9*10 6 CFU / mL, 3.9*10 7 CFU / mL; centrifuge the seven solutions at 5000×g for 5 min, and discard the supernatant; resuspend the precipitate of the seven solutions with 1 mL of phosphate buffered saline, to obtain seven tap water solutions;

[0076] (2) Preparation of test solution

[0077] The ratio fluorescent probe was mixed with seven parts of tap water solution at a volume ratio of 1:1 and incubated for 25 min to obtain seven test solutions;

[0078] (3) Test the liquid to be tested

[0079] The seven samples of the test solution were measured by a red, green and blue three-color imager at an excitation wavelength of 285 nm, and the images were protected from light, and the red, green and blue values ​​of the images were read;

[0080] The ratio between the seven blue values ​​and the green value among the read red, green and blue values ​​was selected as the ordinate, and the logarithm of the bacterial concentration of the test liquid was selected as the abscissa to draw a standard curve, and the correlation coefficient and detection limit were obtained according to the linear regression equation.

[0081] Under the same conditions, uninoculated tap water was used as the blank control test solution, in which the concentration of Listeria monocytogenes was 0 CFU / mL.

[0082] The seven test liquids obtained in Example 2 were respectively subjected to 285nm excitation treatment using a micro-fluorescence spectroscopy device, and the RGB red, green and blue three-color imager (online test colorimeter produced by Admesy of the Netherlands, model Hyperion) was used to analyze the RGB red, green and blue three-color values ​​of the images of the seven test liquids subjected to the excitation treatment; finally, a standard curve was drawn with the ratio of the blue value to the green value as the ordinate and the logarithmic value of the bacterial concentration of the seven test liquids as the abscissa, and the correlation coefficient and the detection limit were obtained based on the linear regression equation.

[0083] Tap water solution:

[0084] See also Figure 1 In A, from left to right ("0", "1", "2", "3", "4", "5", "6", "7") are: detection images with a bacterial concentration of 0 CFU / mL (blank control test solution), bacterial concentration of 3.9*10 1 CFU / mL detection image, bacterial concentration is 3.9*10 2 CFU / mL detection image, bacterial concentration is 3.9*10 3 CFU / mL detection image, bacterial concentration is 3.9*10 4 CFU / mL detection image, bacterial concentration is 3.9*10 5 CFU / mL detection image, bacterial concentration is 3.9*10 6 CFU / mL detection image, bacterial concentration is 3.9*10 7 The detection images of CFU / mL, and the ratios between the blue value and the green value corresponding to the above eight detection images are 2.15, 1.85, 1.68, 1.51, 1.39, 1.28, 1.18, and 1.10 respectively. Figure 1 B in the image is used to analyze the RGB red, green and blue color values ​​of the detection image. Figure 1 The ratio of the blue value to the green value of each detection image in A is taken as the ordinate, and the logarithm of the bacterial concentration corresponding to each detection image is taken as the abscissa. The linear regression equation is y=-0.11995x+1.97527, and the linear fitting coefficient of tap water (r 2 ) was 0.98211, and its detection limit was 6.31 CFU / mL.

[0085] For the tap water solution, when the ratio between the blue value and the green value in the read red, green and blue values ​​is greater than 1.85, Listeria monocytogenes is not detected in the tap water; when the ratio is less than 1.85, Listeria monocytogenes is contained in the tap water. The concentration of Listeria monocytogenes in the tap water solution can be obtained by substituting the ratio into the linear regression equation.

[0086] In this Example 2, the test result of the tap water taken from the tap water pipe of the school toilet was that the concentration of Listeria monocytogenes was 0 CFU / mL.

[0087] Example 3 (the food tested is orange juice)

[0088] The orange juice of Example 3 was purchased from a supermarket on campus. When purchased, the orange juice was stored in a refrigerator at 4° C. The manufacturer was Jiangsu Taikoo Coca-Cola Beverage Co., Ltd. Its brand was “Minute Maid Orange with Pulp”. Its type was orange juice beverage. Its ingredients were water, fruit pulp (orange pulp, grapefruit cysts), white sugar, concentrated orange juice, food additives (citric acid, sodium citrate, β-carotene), edible flavors, and microbial C. The juice content was not less than 10%. The average amount of fruit pulp added was: 3.35 g / 100 ml of orange pulp, 0.55 g / 100 ml of grapefruit cysts, of which 9.8 g of carbohydrates, 22 mg of sodium, and 7.5 mg of vitamin C were present per 100 ml.

[0089] The steps for visual detection of foodborne Listeria monocytogenes using ratiometric fluorescent probes are as follows:

[0090] (1) Preparation of orange juice solution

[0091] Listeria monocytogenes was artificially inoculated into 1 mL of orange juice, and the final concentrations of Listeria monocytogenes were 3.9*10 1 CFU / mL, 3.9*10 2 CFU / mL, 3.9*10 3 CFU / mL, 3.9*10 4 CFU / mL, 3.9*10 5 CFU / mL, 3.9*10 6 CFU / mL, 3.9*10 7 CFU / mL of seven orange juice solutions; centrifuge the seven orange juice solutions at 5000×g for 5 min respectively, and discard the supernatant; resuspend the precipitates of the seven solutions with 1 mL of phosphate buffered saline respectively to obtain seven orange juice solutions.

[0092] (2) Preparation of test solution

[0093] The ratio fluorescent probe was mixed with seven portions of orange juice solution at a volume ratio of 1:1, and incubated for 25 min to obtain seven portions of test solution.

[0094] (3) Test the liquid to be tested

[0095] The seven samples of the test solution were measured by a red, green and blue three-color imager at an excitation wavelength of 285 nm, and the images were protected from light, and the red, green and blue values ​​of the images were read;

[0096] The ratio between the seven blue values ​​and the green value among the read red, green and blue values ​​was selected as the ordinate, and the logarithm of the bacterial concentration of the test liquid was selected as the abscissa to draw a standard curve, and the correlation coefficient and detection limit were obtained according to the linear regression equation.

[0097] Under the same conditions, the uninoculated orange juice solution was used as the blank control test solution, in which the concentration of Listeria monocytogenes was 0 CFU / mL.

[0098] The seven test liquids obtained in Example 3 were respectively subjected to 285nm excitation treatment using a micro-fluorescence spectroscopy device, and the RGB red, green and blue three-color imager (online test colorimeter produced by Admesy of the Netherlands, model Hyperion) was used to analyze the RGB red, green and blue three-color values ​​of the images of the seven test liquids subjected to the excitation treatment; finally, a standard curve was drawn with the ratio of the blue value to the green value as the ordinate and the logarithmic value of the bacterial concentration of the seven test liquids as the abscissa, and the correlation coefficient and the detection limit were obtained based on the linear regression equation.

[0099] Orange juice solution:

[0100] See also Figure 2 In A, from left to right ("0", "1", "2", "3", "4", "5", "6", "7") are: detection images with a bacterial concentration of 0 CFU / mL (blank control test solution), bacterial concentration of 3.9*10 1 CFU / mL detection image, bacterial concentration is 3.9*10 2 CFU / mL detection image, bacterial concentration is 3.9*10 3 CFU / mL detection image, bacterial concentration is 3.9*10 4 CFU / mL detection image, bacterial concentration is 3.9*10 5 CFU / mL detection image, bacterial concentration is 3.9*10 6 CFU / mL detection image, bacterial concentration is 3.9*10 7 CFU / mL detection image, and the ratios between the blue value and the green value corresponding to the above eight detection images are 2.12, 1.91, 1.72, 1.56, 1.42, 1.31, 1.21, and 1.12 respectively. Figure 2 B in the image is used to analyze the RGB red, green and blue color values ​​of the detection image. Figure 2 The ratio between the blue value and the green value of each detection image in A is taken as the ordinate, and the logarithm of the bacterial concentration corresponding to each detection image is taken as the abscissa. The linear regression equation is y=-0.12261x+2.02883. The linear fitting coefficient of orange juice (r 2) was 0.98090, and its detection limit was 8.51 CFU / mL.

[0101] For the orange juice solution, when the ratio between the blue value and the green value in the read red, green and blue values ​​is greater than 1.91, Listeria monocytogenes is not detected in the orange juice; when the ratio is less than 1.91, Listeria monocytogenes is contained in the orange juice, and the concentration of Listeria monocytogenes in the orange juice solution can be obtained by substituting the ratio into the linear regression equation.

[0102] The detection result of the orange juice in this Example 3 was that the concentration of Listeria monocytogenes was 0 CFU / mL.

[0103] Example 4 (the food tested is milk)

[0104] The milk in this embodiment was purchased from the supermarket on campus. When purchased, the milk was stored in a refrigerator at 4°C. The manufacturer is Inner Mongolia Mengniu Dairy (Group) Co., Ltd., its brand is "Mengniu Pure Milk", its type is full-fat sterilized milk, its ingredients are raw cow's milk, and the protein content in every 100g of milk is 3.2g, and the fat content is 4.0g.

[0105] The steps for visual detection of foodborne Listeria monocytogenes using ratiometric fluorescent probes are as follows:

[0106] (1) Preparation of milk solution

[0107] Listeria monocytogenes was artificially inoculated into 1 mL of milk, and the final concentrations of Listeria monocytogenes were 3.9*10 1 CFU / mL, 3.9*10 2 CFU / mL, 3.9*10 3 CFU / mL, 3.9*10 4 CFU / mL, 3.9*10 5 CFU / mL, 3.9*10 6 CFU / mL, 3.9*10 7 CFU / mL; centrifuge the seven solutions at 5000×g for 5 min, and discard the supernatant; resuspend the precipitate of the seven solutions with the supernatant discarded with 1 mL of phosphate buffered saline to obtain seven milk solutions;

[0108] (2) Preparation of test solution

[0109] The ratio fluorescent probe was mixed with seven parts of milk solution at a volume ratio of 1:1 and incubated for 25 min to obtain seven test solutions;

[0110] (3) Test the liquid to be tested

[0111] The seven samples of the test solution were measured by a red, green and blue three-color imager at an excitation wavelength of 285 nm, and the images were protected from light, and the red, green and blue values ​​of the images were read;

[0112] The ratio between the seven blue values ​​and the green value among the read red, green and blue values ​​was selected as the ordinate, and the logarithm of the bacterial concentration of the test liquid was selected as the abscissa to draw a standard curve, and the correlation coefficient and detection limit were obtained according to the linear regression equation.

[0113] Under the same conditions, the uninoculated milk solution was used as the blank control test solution, in which the concentration of Listeria monocytogenes was 0 CFU / mL.

[0114] The seven test liquids obtained in Example 4 were respectively subjected to 285 nm excitation treatment using a micro-fluorescence spectroscopy device, and the RGB red, green and blue three-color imager (online test colorimeter produced by Admesy of the Netherlands, model Hyperion) was used to analyze the RGB red, green and blue three-color values ​​of the images of the seven test liquids subjected to the excitation treatment; finally, a standard curve was drawn with the ratio of the blue value to the green value as the ordinate and the logarithmic value of the bacterial concentration of the seven test liquids as the abscissa, and the correlation coefficient and the detection limit were obtained based on the linear regression equation.

[0115] See also Figure 3 In A, from left to right ("0", "1", "2", "3", "4", "5", "6", "7") are: detection images with a bacterial concentration of 0 CFU / mL (blank control test solution), bacterial concentration of 3.9*10 1 CFU / mL detection image, bacterial concentration is 3.9*10 2 CFU / mL detection image, bacterial concentration is 3.9*10 3 CFU / mL detection image, bacterial concentration is 3.9*10 4 CFU / mL detection image, bacterial concentration is 3.9*10 5 CFU / mL detection image, bacterial concentration is 3.9*10 6 CFU / mL detection image, bacterial concentration is 3.9*10 7 CFU / mL detection image, and the ratios between the blue value and the green value corresponding to the above eight detection images are 2.11, 1.90, 1.72, 1.56, 1.43, 1.32, 1.22, and 1.14 respectively; see Figure 3 B in the image is used to analyze the RGB red, green and blue color values ​​of the detection image. Figure 3The ratio between the blue value and the green value of each detection image in A is taken as the ordinate, and the logarithm of the bacterial concentration corresponding to each detection image is taken as the abscissa. The linear regression equation is y=-0.12302x+2.03473, and the linear fitting coefficient of milk (r 2 ) was 0.98242, and its detection limit was 8.93 CFU / mL.

[0116] For the milk solution, when the ratio between the blue value and the green value in the read red, green and blue values ​​is greater than 1.90, Listeria monocytogenes is not detected in the milk; when the ratio is less than 1.90, Listeria monocytogenes is contained in the milk. The concentration of Listeria monocytogenes in the milk solution can be obtained by substituting the ratio into the linear regression equation.

[0117] The test result of the milk in this Example 4 was that the concentration of Listeria monocytogenes was 0 CFU / mL.

[0118] Example 5 (the food tested is cooked beef)

[0119] The cooked beef is purchased from the school canteen. When purchased, it is stored in the canteen window (cold dish window) and the temperature of the cooked beef is 26℃.

[0120] The steps for visual detection of foodborne Listeria monocytogenes using ratiometric fluorescent probes are as follows:

[0121] (1) Preparation of cooked beef solution

[0122] The cooked beef was inoculated with Listeria monocytogenes at a volume of 3.9*10 8 CFU / g, and let it stand for 2 hours at room temperature to obtain inoculated cooked beef; cut the inoculated cooked beef into small pieces; in a sterilization bag, mix 25g of the inoculated cooked beef cut into small pieces with 25mL of phosphate buffered saline solution, homogenize for 2 minutes to prepare a phosphate buffered saline solution mixture, let the mixture stand for 5 minutes, and take the supernatant; by adding phosphate buffered saline solution, the final concentration of Listeria monocytogenes in the supernatant was adjusted to 3.9*10 1 CFU / mL, 3.9*10 2 CFU / mL, 3.9*10 3 CFU / mL, 3.9*10 4 CFU / mL, 3.9*10 5 CFU / mL, 3.9*10 6 CFU / mL, 3.9*10 7 CFU / mL seven parts solution, that is, seven parts cooked beef solution.

[0123] (2) Preparation of test solution

[0124] The ratio fluorescent probe was mixed with seven parts of tap water solution at a volume ratio of 1:1 and incubated for 25 min to obtain seven test solutions;

[0125] (3) Test the liquid to be tested

[0126] The seven samples of the test solution were measured by a red, green and blue three-color imager at an excitation wavelength of 285 nm, and the images were protected from light, and the red, green and blue values ​​of the images were read;

[0127] The ratio between the seven blue values ​​and the green value among the read red, green and blue values ​​was selected as the ordinate, and the logarithm of the bacterial concentration of the test liquid was selected as the abscissa to draw a standard curve, and the correlation coefficient and detection limit were obtained according to the linear regression equation.

[0128] Under the same conditions, the uninoculated cooked beef solution was used as the blank control test solution, in which the concentration of Listeria monocytogenes was 0 CFU / mL.

[0129] The seven test liquids obtained in Example 5 were respectively subjected to 285nm excitation treatment using a micro-fluorescence spectroscopy device, and the RGB red, green and blue three-color imager (online test colorimeter produced by Admesy of the Netherlands, model Hyperion) was used to analyze the RGB red, green and blue three-color values ​​of the images of the seven test liquids subjected to the excitation treatment; finally, a standard curve was drawn with the ratio of the blue value to the green value as the ordinate and the logarithmic value of the bacterial concentration of the seven test liquids as the abscissa, and the correlation coefficient and the detection limit were obtained based on the linear regression equation.

[0130] Cooked Beef Solution:

[0131] See also Figure 4 In A, from left to right ("0", "1", "2", "3", "4", "5", "6", "7") are: detection images with a bacterial concentration of 0 CFU / mL (blank control test solution), bacterial concentration of 3.9*10 1 CFU / mL detection image, bacterial concentration is 3.9*10 2 CFU / mL detection image, bacterial concentration is 3.9*10 3 CFU / mL detection image, bacterial concentration is 3.9*10 4 CFU / mL detection image, bacterial concentration is 3.9*10 5 CFU / mL detection image, bacterial concentration is 3.9*10 6 CFU / mL detection image, bacterial concentration is 3.9*10 7CFU / mL detection image, and the ratios between the blue value and the green value corresponding to the above eight detection images are 2.09, 1.97, 1.58, 1.42, 1.31, 1.20, 1.11, and 1.58 respectively; see Figure 4 B in the image is used to analyze the RGB red, green and blue color values ​​of the detection image. Figure 4 The ratio of the blue value to the green value of each detection image in A is taken as the ordinate, and the logarithm of the bacterial concentration corresponding to each detection image is taken as the abscissa. The linear regression equation is y=-0.11452x+1.85036. The linear fitting coefficient of cooked beef (r 2 ) was 0.98945, and its detection limit was 15.09 CFU / mL.

[0132] For the cooked beef solution, when the ratio between the blue value and the green value in the read red, green and blue values ​​is greater than 1.97, Listeria monocytogenes is not detected in the cooked beef; when the ratio is less than 1.97, Listeria monocytogenes is contained in the cooked beef. The concentration of Listeria monocytogenes in the cooked beef solution can be obtained by substituting the ratio into the linear regression equation.

[0133] The detection result of the cooked beef in Example 5 was that the concentration of Listeria monocytogenes was 0 CFU / mL.

[0134] Example 6 (the food tested is lettuce)

[0135] The lettuce is purchased from the school cafeteria and stored in the cafeteria window (Malatang vegetable window) when purchased. The temperature of the lettuce is 4℃.

[0136] The steps for visual detection of foodborne Listeria monocytogenes using ratiometric fluorescent probes are as follows:

[0137] (1) Preparation of lettuce solution

[0138] Listeria monocytogenes was inoculated into lettuce at an inoculation rate of 3.9*10 8 CFU / g, and allowed to stand at room temperature for 2 hours to obtain inoculated lettuce; the inoculated lettuce was cut into pieces; 25g of the inoculated lettuce cut into pieces was mixed with 25mL of phosphate buffered saline in a sterilization bag, and homogenized for 2 minutes to obtain a phosphate buffered saline mixture, the mixture was allowed to stand for 5 minutes, and the supernatant was taken; the final concentration of Listeria monocytogenes in the supernatant was adjusted to 3.9*10 1 CFU / mL, 3.9*10 2 CFU / mL, 3.9*10 3 CFU / mL, 3.9*10 4 CFU / mL, 3.9*10 5 CFU / mL, 3.9*106 CFU / mL, 3.9*10 7 CFU / mL seven parts solution, that is, seven parts lettuce solution;

[0139] (2) Preparation of test solution

[0140] The ratio fluorescent probe was mixed with seven parts of tap water solution at a volume ratio of 1:1 and incubated for 25 min to obtain seven test solutions;

[0141] (3) Test the liquid to be tested

[0142] The seven samples of the test solution were measured by a red, green and blue three-color imager at an excitation wavelength of 285 nm, and the images were protected from light, and the red, green and blue values ​​of the images were read;

[0143] The ratio between the seven blue values ​​and the green value among the read red, green and blue values ​​was selected as the ordinate, and the logarithm of the bacterial concentration of the test liquid was selected as the abscissa to draw a standard curve, and the correlation coefficient and detection limit were obtained according to the linear regression equation.

[0144] Under the same conditions, the non-inoculated lettuce solution was used as the blank control test solution, in which the concentration of Listeria monocytogenes was 0 CFU / mL.

[0145] The seven test liquids obtained in Example 6 were respectively subjected to 285 nm excitation treatment using a micro-fluorescence spectroscopy device, and the RGB red, green and blue three-color imager (online test colorimeter produced by Admesy of the Netherlands, model Hyperion) was used to analyze the RGB red, green and blue three-color values ​​of the images of the seven test liquids subjected to the excitation treatment; finally, a standard curve was drawn with the ratio of the blue value to the green value as the ordinate and the logarithmic value of the bacterial concentration of the seven test liquids as the abscissa, and the correlation coefficient and the detection limit were obtained based on the linear regression equation.

[0146] Cooked Beef Solution:

[0147] See also Figure 5 In A, from left to right ("0", "1", "2", "3", "4", "5", "6", "7") are: detection images with a bacterial concentration of 0 CFU / mL (blank control test solution), bacterial concentration of 3.9*10 1 CFU / mL detection image, bacterial concentration is 3.9*10 2 CFU / mL detection image, bacterial concentration is 3.9*10 3 CFU / mL detection image, bacterial concentration is 3.9*10 4 CFU / mL detection image, bacterial concentration is 3.9*10 5CFU / mL detection image, bacterial concentration is 3.9*10 6 CFU / mL detection image, bacterial concentration is 3.9*10 7 CFU / mL detection image, and the ratios between the blue value and the green value corresponding to the above eight detection images are 2.17, 2.04, 1.83, 1.65, 1.51, 1.38, 1.28, and 1.19 respectively; see Figure 5 B in the image is used to analyze the RGB red, green and blue color values ​​of the detection image. Figure 5 The ratio of the blue value to the green value of each detection image in A is taken as the ordinate, and the logarithm of the bacterial concentration corresponding to each detection image is taken as the abscissa. The linear regression equation is y=-0.13133x+2.15409, and the correlation coefficient (r 2 ) was 0.97770, and its detection limit was 9.60CFU / mL.

[0148] For the lettuce solution, when the ratio between the blue value and the green value in the read red, green and blue values ​​is greater than 2.04, Listeria monocytogenes is not detected in the lettuce; when the ratio is less than 2.04, Listeria monocytogenes is contained in the lettuce, and the concentration of Listeria monocytogenes in the lettuce solution can be obtained by substituting the ratio into the linear regression equation.

[0149] The detection result of the lettuce in Example 6 was that the concentration of Listeria monocytogenes was 0 CFU / mL.

[0150] Example 7

[0151] To increase the detection of real food samples, LB broth samples were selected for the new test:

[0152] It should be noted that in order to verify whether the standard curve established in accordance with Example 2 (the standard curve of tap water solution is selected) can be effectively applied to the determination under the new interference environment (LB broth), the difference between the number of bacteria detected by the probe in the artificial quantitative infection and LB broth samples is respectively carried out to find out that even if there is no time to establish a standard curve in the future when encountering unknown food samples, the visual ratio fluorescent probe established in this application can be effectively detected under the influence of the nutrients of the culture medium.

[0153] (1) Preparation of LB broth solution

[0154] Listeria monocytogenes was artificially inoculated in 1 mL of LB broth to obtain a final concentration of 3.9*10 6 CFU / mL; centrifuge one solution at 5000×g for 5 min and discard the supernatant; resuspend the precipitate in 1 mL of phosphate buffered saline to obtain an LB broth solution.

[0155] (2) Preparation of test solution

[0156] The ratio fluorescent probe was mixed with one portion of LB broth solution at a volume ratio of 1:1 and incubated for 25 min to obtain one portion of the test solution.

[0157] (3) Test the liquid to be tested

[0158] A portion of the test liquid is measured by a red, green and blue three-color imager under an excitation wavelength of 285 nm to generate an image protected from light, and the red, green and blue values ​​of the image are read;

[0159] The ratio between the blue value and the green value in the read red, green and blue values ​​is selected and substituted into the linear regression equation obtained in Example 2 to obtain its concentration value.

[0160] A portion of the test liquid obtained in Example 7 was subjected to 285nm excitation treatment using a micro-fluorescence spectroscopy device, and an RGB red, green and blue three-color imager (an online test colorimeter produced by Admesy of the Netherlands, model Hyperion) was used to analyze the RGB red, green and blue color values ​​of the image of the excited test liquid; finally, the ratio between the blue value and the green value was calculated and substituted into the linear regression equation obtained in Example 2 to obtain its concentration value.

[0161] LB broth solution:

[0162] The test results show that the concentration of Listeria monocytogenes in the LB broth artificially inoculated with Listeria monocytogenes was calculated to be 3.83*10 6 CFU / mL, almost the same as its actual concentration (3.9*10 6 CFU / mL), it can be seen that even if the corresponding standard curve and linear regression equation in the LB broth sample environment are not established, the standard curve and linear regression equation established in the existing tap water environment can still have good versatility. It can be seen that the method of the present invention has good versatility and can be quickly applied to different sample detections.

[0163] It will be easily understood by those skilled in the art that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for visual detection of foodborne Listeria monocytogenes using a ratiometric fluorescent probe, characterized in that: The steps are as follows: (1) Preparation of food solution Artificially inoculating Listeria monocytogenes into the food to be tested to prepare a food solution; the food to be tested is tap water, orange juice, milk, cooked beef, and lettuce, and the food solution is tap water solution, orange juice solution, milk solution, cooked beef solution, and lettuce solution; (2) Preparation of test solution Mix the ratio fluorescent probe with the food solution obtained in step (1) at a volume ratio of 1:1, and incubate for 25 minutes to obtain a test solution; The ratio fluorescent probe is prepared by uniformly mixing a first fluorescent probe as an energy donor and a second fluorescent probe as an energy acceptor in a volume ratio of 1:1; The first fluorescent probe is vancomycin-modified carbon quantum dots; The second fluorescent probe is aptamer-modified silicon nanoparticles; The DNA sequence of the aptamer is shown in SEQ ID No: 1, and the 5' end of the aptamer is modified with an amino group; In the ratiometric fluorescent probe, the fluorescence emission wavelength of the vancomycin-modified carbon quantum dots is 458 nm, and the fluorescence emission wavelength of the aptamer-modified silicon nanoparticles is 488 nm; (3) Test the liquid to be tested The liquid to be tested obtained in step (2) is measured by a red, green and blue three-color imager under an excitation wavelength of 285 nm to generate an image of the liquid to be tested in the dark, and the red, green and blue values ​​of the image are read; The ratio between the blue value and the green value in the red, green and blue values ​​is selected as the ordinate, and the logarithm of the bacterial concentration of the test solution is selected as the abscissa to draw a standard curve, and the correlation coefficient and the detection limit are obtained according to the linear regression equation; The linear fitting coefficient of the tap water is 0.98211, and the detection limit is 6.31 CFU / mL. For the tap water solution, when the ratio between the blue value and the green value in the red, green and blue values ​​read is greater than 1.85, Listeria monocytogenes is not detected in the tap water; when the ratio is less than 1.85, Listeria monocytogenes is contained in the tap water, and the concentration of Listeria monocytogenes in the tap water solution can be obtained by substituting the ratio into the linear regression equation; The linear fitting coefficient of the orange juice is 0.98090, and the detection limit is 8.51 CFU / mL. For the orange juice solution, when the ratio between the blue value and the green value in the red, green and blue values ​​read is greater than 1.91, Listeria monocytogenes is not detected in the orange juice; when the ratio is less than 1.91, Listeria monocytogenes is contained in the orange juice, and the concentration of Listeria monocytogenes in the orange juice solution can be obtained by substituting the ratio into the linear regression equation. The linear fitting coefficient of the milk is 0.98242, and the detection limit is 8.93 CFU / mL. For the milk solution, when the ratio between the blue value and the green value in the red, green and blue values ​​read is greater than 1.90, Listeria monocytogenes is not detected in the milk; when the ratio is less than 1.90, Listeria monocytogenes is contained in the milk, and the concentration of Listeria monocytogenes in the milk solution can be obtained by substituting the ratio into the linear regression equation. The linear fitting coefficient of the cooked beef is 0.98945, and the detection limit is 15.09 CFU / mL; for the cooked beef solution, when the ratio between the blue value and the green value in the red, green and blue values ​​read is greater than 1.97, Listeria monocytogenes is not detected in the cooked beef; When the ratio is less than 1.97, the cooked beef contains Listeria monocytogenes, and the concentration of Listeria monocytogenes in the cooked beef solution can be obtained by substituting the ratio into the linear regression equation; The linear fitting coefficient of the lettuce is 0.97770, and the detection limit is 9.60 CFU / mL; for the milk solution, when the ratio between the blue value and the green value in the red, green and blue values ​​read is greater than 2.03, Listeria monocytogenes is not detected in the milk; when the ratio is less than 2.03, Listeria monocytogenes is contained in the milk, and the concentration of Listeria monocytogenes in the milk solution can be obtained by substituting the ratio into the linear regression equation; In step (2), the preparation operation of the ratiometric fluorescent probe is as follows: (2.1) Preparation of activation mixture Mix 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide in a volume ratio of 1:1 to obtain an activated mixed solution; (2.2) Preparation of the first fluorescent probe Guanidine hydrochloride and ultrapure water were mixed and dissolved at a mass volume ratio of 12 mg: 2 mL to obtain a mixed solution; the mixed solution was transferred to a reactor, the reaction temperature was 200°C, the reaction time was 8 hours, and the reaction solution was cooled to room temperature to obtain a reaction solution; the reaction solution was centrifuged at a speed of 8000 rpm and a temperature of 4°C for 20 minutes, and the supernatant was collected; the supernatant was mixed with the activation mixed solution at a volume ratio of 2:1, and incubated at room temperature for 25 minutes; a 50 uM vancomycin solution was added, the amount of the vancomycin solution added was 0.006 times the volume of the supernatant, and the reaction was incubated on a shaker at a speed of 180 rpm and room temperature for 2.5 hours to obtain a first fluorescent probe; (2.3) Preparation of the second fluorescent probe A sodium ascorbate solution with a molar concentration of 100 mM and 3-aminopropyltrimethoxysilane are mixed in a volume ratio of 1:1, and eight times the volume of ultrapure water is added and stirred for 80 minutes; dialyzed with a 500Da dialysis bag for 18 hours to obtain a retentate, and the retentate is diluted fifteen times; the retentate is mixed with the activation mixture in a volume ratio of 2:1, and incubated at room temperature for 30 minutes; an aptamer solution with a concentration of 100 uM is added, and the amount of the aptamer solution added is 0.03 times the volume of the retentate; at a rotation speed of 180 rpm and room temperature, the shaker is incubated for 2.5 hours to obtain a second fluorescent probe; (2.4) Preparation of ratiometric fluorescent probe The first fluorescent probe and the second fluorescent probe are mixed at a volume ratio of 1:1 to prepare a ratio fluorescent probe; When the fluorescence emission wavelength of the carbon quantum dots shifts from 452nm to 458nm, it indicates that vancomycin in the ratio fluorescence probe is successfully modified onto the carbon quantum dots. When the fluorescence emission wavelength of the silicon nanoparticles shifts from 500nm to 488nm, it indicates that the aptamer in the ratio fluorescence probe is successfully modified onto the silicon nanoparticles.

2. The method for visual detection of foodborne Listeria monocytogenes using a ratiometric fluorescent probe according to claim 1, characterized in that: In step (1), the steps for preparing the tap water solution, orange juice solution or milk solution are as follows: Listeria monocytogenes was artificially inoculated into 1 mL of tap water, orange juice or milk, and the final concentration of Listeria monocytogenes was 3.9*10 1 CFU / mL, 3.9*10 2 CFU / mL, 3.9*10 3 CFU / mL, 3.9*10 4 CFU / mL, 3.9*10 5 CFU / mL, 3.9*10 6 CFU / mL, 3.9*10 7 CFU / mL; centrifuge the seven solutions at 5000×g for 5 min respectively and discard the supernatant; resuspend the precipitates of the seven solutions with the supernatant discarded in 1 mL of phosphate buffered saline respectively to obtain seven tap water solutions or orange juice solutions or milk solutions.

3. The method for visual detection of foodborne Listeria monocytogenes using a ratiometric fluorescent probe according to claim 1, characterized in that: In step (1), the steps for preparing the cooked beef solution or the lettuce solution are as follows: (1.1) Inoculate cooked beef or lettuce with Listeria monocytogenes at an inoculum of 3.9*10 8 CFU / g, after standing for 2 h at room temperature, inoculated cooked beef or inoculated lettuce were obtained; (1.2) Cut the inoculated cooked beef into small pieces or cut the inoculated lettuce into pieces; (1.3) In a sterilization bag, mix 25 g of inoculated cooked beef cut into small pieces or 25 g of inoculated lettuce cut into pieces with 25 mL of phosphate buffered saline solution and homogenize for 2 minutes to prepare a phosphate buffered saline mixture. Let the mixture stand for 5 minutes and take the supernatant. The final concentration of Listeria monocytogenes in the supernatant was adjusted to 3.9*10 1 CFU / mL, 3.9*10 2 CFU / mL, 3.9*10 3 CFU / mL, 3.9*10 4 CFU / mL, 3.9*10 5 CFU / mL, 3.9*10 6 CFU / mL, 3.9*10 7 CFU / mL is seven parts solution, that is, seven parts cooked beef solution or seven parts lettuce solution.

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